Sulfotransferase (ST) catalyzed sulfation is an important pathway in the metabolism of hydroxyl (aryl or alkyl) containing drugs, carcinogens, xenobiotics and endogenous compounds. Sulfation of drugs and xenobiotics is mostly associated with detoxification by which a relatively hydrophobic compound is biotransformed into a more water-soluble sulfuric ester that is readily excreted. However, there are numerous important exceptions wherein the formation of chemically reactive sulfuric esters is an essential step in metabolic pathways leading to toxic or carcinogenic bioactivation. Detoxification or bioactivation is highly dependent upon the electrophilic reactivity of the individual sulfuric ester products formed. The long-term objective of the proposed research is to define the structural characteristics of the active sites of human STs and to investigate the catalytic mechanism of the sulfation reaction catalyzed by STs. An enzyme's catalytic mechanism and substrate specificity are determined by the micro-environment produced by specific amino acid residues localized within the active site and by the three dimensional structure of the protein molecule. According to our previous studies and reports in the literature, we hypothesize that the co-substrate PAPS binding site is composed of three highly conserved regions and other elements, and the substrate specificity of different STs is dictated by amino acid residues in several variable regions. Specific investigations to be carried out in this proposal are as follows: 1. Use photoaffinity probes to identify amino acid residues in both the substrate- binding site and PAPS binding site of human liver phenol- sulfating ST (P-PST-1), dehydroepiandrosterone ST (DHEA-ST), and estrogen ST (EST). 2. Use amino acid modification reagents to identify the essential amino acid residues in the catalytic active site of STs. 3. Mutagenesis of essential amino acids and/or functional domains of human STs. 4.Computational molecular structure studies of human STs. The computational molecular structure studies, combining the experimental results from specific aims 1-3, will help us to understand the catalytic mechanism and differing substrate specificity of human STs. The information on human ST active site structure, substrate specificity, and catalytic mechanism should have important implications for the prediction of biotransformation pathways and inter-individual differences in metabolism of endogenous compounds, xenobiotics, and carcinogens. The characterization of the active sites of STs will also help to find mechanism-based specific inhibitors/activators for use in studying the physiological functions of STs and designing new drugs.